肾癌ACHN细胞exosome对自身细胞增殖和凋亡的影响

Effects of renal carcinoma cell line ACHN-derived exosomes on ACHN cell proliferation and apoptosis

目的探讨肾癌ACHN细胞来源的exosome对肾癌ACHN细胞自身增殖和凋亡的影响及机制。方法用超滤和蔗糖重水密度梯度超速离心法分离纯化肾癌ACHN细胞分泌的exosome;采用CCK-8法评价exosome对肾癌ACHN细胞增殖的影响;Annexin V-FITC/PI双染色流式细胞术检测细胞凋亡的变化;RT-PCR和Western blotting检测CyclinD1、caspase-3 mRNA和蛋白的表达;Western blotting检测p-Akt、p-ERK1/2的变化。结果成功使用超滤和蔗糖重水密度梯度超速离心法分离纯化肾癌ACHN细胞分泌的exosome。exosome可促进肾癌ACHN细胞增殖,抑制其凋亡;在此过程中,CyclinD1 mRNA和蛋白的表达均上调,而caspase-3 mRNA表达无明显变化,caspase-3蛋白表达下调;同时p-Akt和p-ERK1/2的表达也上调。结论 exosome可促进肾癌ACHN细胞生长和增殖,抑制细胞凋亡。筛除肾癌微环境中的exosome或抑制其功能,可能成为肾癌治疗的有效新方法。

Objective To investigate the effects of exosomes derived from renal cancer cell line ACHN on the proliferation and apoptosis of ACHN cells and explore the mechanism.Methods Exosomes derived from ACHN cells were separated and purified by ultrafiltration and sucrose gradient centrifugation.The effects of the exosomes on the proliferation and apoptosis of ACHN cells were analyzed with CCK-8 assay and flow cytometry,respectively.The changes of mRNA and protein expressions of cyclin D1,caspase-3 were examined using RT-PCR and Western blotting,and the changes in the protein expression of p-Akt and p-ERK1/2 were detected with Western blotting.Results Exosomes were successfully purified by ultrafiltration and sucrose gradient centrifugation.Compared with the control cells,ACHN cells treated with the exosomes showed enhanced proliferative activity with suppressed cell apoptosis.Exosomes treatment upregulated cyclinD1 mRNA and protein expression,down-regulated caspase-3 protein expression without affecting caspase-3 mRNA expression,and upregulated the expression of p-Akt and p-ERK1/2.Conclusion Exosomes can promote the growth and proliferation and inhibit the apoptosis of renal cancer cell line ACHN.Removal of the exosomes from the microenvironment of renal cancer or inhibition of its function can be new strategies for treatment of renal cancer.